Abstract
Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.
Highlights
Immune protection relies on a functional adaptive immune system
There, cytotoxic CD8+ T cells are crucial for the elimination of intracellular pathogens, while the production of highly specific antibodies from B cells and coordination of innate immunity is impossible without CD4+ T cells
The group of from Alexander Marson showed that homology-directed repair (HDR) efficiency can be twoto four-fold improved by adding the short truncated Cas9 target sequences to homology arms of doublestranded DNA (ds-DNA) templates
Summary
Immune protection relies on a functional adaptive immune system. Its important parts are T cells characterized by the expression of a unique clone-specific cell-surface protein complex, the T-cell receptor (TCR). One of the first events during HDR is the activation and recruitment of phosphorylases, such as ataxia-telangiectasia-mutated protein (ATM) and Rad3-related kinase (ATR) to the site of damage [64] These enzymes phosphorylate several proteins, including the DNA-stabilizing histone H2AX on serine 139 [65,66,67], which seems to play an important role in the recruitment of additional repair and signaling molecules to the DSB [68]. RPA is replaced by DNA repair protein RAD51 homolog 1 (RAD51) that mediates invasion of ss-DNA filament on the homologous DNA template, usually the complementary strand of the sister chromatid DNA duplex, which is is used to synthesize the missing genomic parts [70,71] This mechanism of HDR is exploited for repairing CRISPR/Cas-induced DSBs. Targeted delivery of synthetic DNA provides an artificial HDR template that can be used to insert genetic information by the cells’ own repair mechanisms. For successful application of CRISPR/Cas-mediated genome editing by using either the NHEJ or HDR pathway, several practical aspects appear to be critical
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